A Sequence Listing has also been included herein in accordance with the provisions of 37 C.F.R. xc2xa71.821 et seq. To the extent any discrepancy exists between the Specification Figures and the Sequence Listing, the Specification or Figures should be considered to be the primary document.
The present invention relates generally to compositions and methods for producing recombinant adeno-associated virus (rAAV) vectors. More specifically, the present invention relates to packaging cell lines and methods for making and using them. Moreover, the rAAV packaging cell lines of the present invention are used to produce high-titer rAAV, that is free of replication-competent AAV and that are suitable for a wide range of applications including ex vivo and in vivo gene therapy as well as in vitro recombinant protein production.
Adeno-associated virus (AAV) is a ubiquitous single stranded DNA parvovirus capable of infecting a wide range of cell types from a variety of different species. Under normal physiological conditions, AAV enters the host cell where it is transported to the cell nucleus. Once inside the cell nucleus, the viral capsid is removed and the viral DNA is stably integrated into the host chromosome. After integration, AAV remains dormant and is generally incapable of self-replication. However, AAV replication can be induce when the cell containing the latent AAV DNA is co-infected with either an adenovirus or a member of the hepresviradae, including herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein Barr virus (EBV) or Vaccina Virus and pseudorabies virus (Berns, K. I. Parvoviridae: The Viruses and Their Replication. In: Fields, B. N. ed. Virology. Philadelphia. Lippincott-Raven 1996 Third Edition Vol. 2 2181-2192.) These so-called xe2x80x9chelper virusesxe2x80x9d provide AAV the necessary helper functions required to rescue and activate the AAV genome and initiate transcription.
Gene therapy, which provides a method for altering the genetic repertoire of cells for a therapeutic benefit has shown promise for treating or preventing a number of diseases. For example, such therapies are now being tested in clinical trials for a range of hereditary (e.g., ADA deficiency, familial hypercholesterolemia, and cystic fibrosis) and acquired (e.g., cancer, viral infection) diseases (Crystal, Science 270:404-410, 1995). Furthermore, gene therapy has shown promise for a variety of vaccine applications.
Many different types of vectors, principally viral vectors, can be utilized for a variety of gene therapy applications, including for example, viral vectors derived from retroviruses, adenoviruses, poxviruses, herpes viruses, and adeno-associated viruses (see Jolly, Cancer Gene Therapy 1:51-64, 1994). One difficulty, however, for present viral-based vectors (and for adeno-associated viral vectors in particular), is that large quantities of viral particles are difficult to produce in a cost-efficient commercial setting.
Data from animal experiments suggest that recombinant AAV (rAAV) may be useful in delivering genes to treat a number of diseases including hemophilia A and B, Gauche""s disease, Parkinson""s disease and retinitis pigmentosa. Despite this experimental success, there is only one human trial in progress with an AAV vector compared to hundreds already conducted with retrovirus or adenovirus vectors. One reason for this disparity is that there has been less development of AAV based vectors, and this in turn reflects the amount of attention that the basic biology each virus group has received. A second and more practical reason is the difficulty in obtaining the amount of rAAV needed for clinical trials, let alone a medical product. The current trial uses transient transfection to manufacture material, a procedure suited to the lab bench but not particularly friendly to a manufacturing suite. As an alternative, cell line technology is more easily scaled and far less likely to generate replication competent virus.
So far the approach for making recombinant AAV producer cell lines employs the techniques used for retrovirus vectorsxe2x80x94remove the origins and packaging sequences from the viral genes and select for stable integration by co-transfecting the remainder with a resistance marker. For AAV, the origins and packaging sequence are found in the inverted terminal repeats (ITR""s). This approach has proven far less successful with AAV than retrovirus. Of the resulting clones, only a few contain intact AAV genomes, and even fewer are capable of making vector particles. Out of these, only an extremely rare clone makes a useful amount of vector (Gao, G. P et al. High-Titer Adenoassociated Viral Vectors from a Rep/Cap Cell Line and Hybrid Shuttle Virus. Human Gene Therapy. 9:2353-62).
AAV efficiently establishes latent infections in the absence of helper virus (Berns, K. I. et al. Adeno-associated virus Latent Infections. In: MayBWJ et al. eds. Virus Persistence. Cambridge: Cambridge University press. 1982; 249.). This natural pathway is tantalizing to anyone trying to create new rAAV packaging technology since such latently infected cells appear to be stable for many generations, and in contrast to transfected cells, virtually all the latently infected cells can be activated to make up to 106 particles of AAV(Berns, K. I. et al. Adeno-associated virus Latent Infections. In: May BWJ et al. eds. Virus Persistence. Cambridge: Cambridge University press. 1982; 249). Clearly the differences between wild type AAV and current producer cell lines are critical. One important difference is that AAV integrates into a limited number of specific sites in human DNA as opposed to random integration by transfection and selection (Cheung A-M et al. 1980. J. Virol. 33:739). Specific integration appears to require three components: AAV ITR""s containing rep-binding sites, chromosomal DNA with rep binding sites, and rep protein (Chapman MS et al. 1993. Virol. 194:491). The silent state of latent virus and its efficient activation by helper virus maybe properties of the chromosomal location of the latent viral genomes. However, data indicates that at least for activation of AAV expression the ITR""s are also a critical component (Im, DS et al. 1989. J. Virol. 63:3095).
The present invention discloses novel compositions and methods for generating recombinant AAV vectors, and further provides other related advantages.
Briefly stated, the present invention provides compositions and methods for generating recombinant AAV vectors. Specifically, the present invention provides pharmaceutical preparations of rAAV suitable for use in ex vivo and in vivo gene therapy as well as in vitro recombinant antigen production. Generally, the present invention provides high-titer rAAV suspensions that are produced in eukaryotic cells. The rAAV suspensions are free from replication-competent rAAV, wild type AAV. This is achieved by infecting a suitable host eukaryotic cell using a first recombinant AAV vector having a first site specific recombination locus inserted between the 5xe2x80x2 inverted terminal repeat (ITR) sequence and the rep gene and a second site specific recombination locus inserted between the 3xe2x80x2 ITR and the cap gene.
Next, the host cell infected with the first rAAV is infected with a second rAAV having a gene of interest substituted for the rep and cap regions of the AAV genome. The two rAAVs may be used to infect the host cell simultaneously, or sequentially. Recombinant AAV infectious particle production and packaging is induced by infecting the host cell containing the first and second rAAV genomes using a wild type helper virus and two helper virus recombinant variants. The first recombinant variant helper virus expresses a recombinase gene (Cre) and the second recombinant variant helper virus has a site-specific recombination locus genome insert. It is understood that the helper virus infection of the eukaryotic host cell may proceed in any order, or may be performed simultaneously.
In another embodiment of the present invention rAAV production and packaging is induced using a wild type helper virus and a single recombinant helper virus having both a site specific recombination locus and a gene encoding for Cre recombinase.
It yet another embodiment of the present invention the recombinant helper virus has a site specific recombination locus, a Cre recombinase gene and all necessary helper genes. In this embodiment only a single helper virus is necessary to induce the production of replication incompetent recombinant AAV particles.
Within one aspect of the present invention recombinant adeno-associated virus are provided comprising 5xe2x80x2 AAV ITR, a first site specific recombination locus, rep and cap genes, and a second site specific recombination locus which is capable of recombining with the first site specific recombination locus. Within preferred embodiments, the virus further comprise a 3xe2x80x2 AAV ITR. Representative site-specific recombination loci are loxP and FRT. Also provided are plasmids that comprise a DNA sequence of the aforementioned viruses. In another embodiment of the present invention a recombinant retrovirus is provided having an 5xe2x80x2LTR, a packaging signal, an AAV rep region, an AAV cap region and a 5xe2x80x2 LTR. Other recombinant retrovirus variations are also possible which include, but are not limited to, inverting the AAV rep and cap genes relative to each other, adding selection markers and truncating the LTRs.
Within other embodiments of the invention, methods are provided for producing recombinant AAV vector particles; comprising the steps of (a) introducing into a host cell (i) pfloxAAV, (ii) a recombinant AAV vector encoding plasmid, and (iii) a plasmid encoding a member of the herpesviridae (e.g., herpesvirus or cytomegalovirus), Epstein-Barr virus, or adenovirus, which supplies necessary helper functions, or, a virus or viral vector which encodes such functions, in order to produce flox AAV particles and recombinant AAV particles; and (b) introducing into a second host cell (i) the recombinant AAV particles and flox AAV particles of (a), (ii) a vector which directs the expression of Cre, and (iii) a vector which directs the expression of herpesvirus, CMV, or adenovirus helper functions, such that said recombinant AAV vector particles are produced. Within further embodiments, a vector which directs the expression of cap may also be introduced into the second host cell. Within certain embodiments, the second host cell does not produce E1A. Further, as should be readily evident a variety of vectors or particles other than pflox AAV or flox AAV particles may be utilized in the context of the present invention, including for example, vectors or particles that have at least one site specific recombination locus as discussed above.
Within further embodiments of the invention plasmids are provided which encode a member of the herpesviridae (e.g., herpesvirus or cytomegalovirus), vaccinia virus, Epstein-Barr virus, or adenovirus, which supplies necessary helper functions (in order to produce flox AAV particles and recombinant AAV particles), and which also have a pair of site specific recombination locus (e.g., loxP sequences).
Within other aspects of the present invention, host cells are provided which comprise an integrated DNA sequence of the recombinant adeno-associated virus described herein. Within further embodiments, the host cells further comprise a recombinant AAV vector.
Within further aspects of the present invention, circular DNA is provided, comprising rep and cap genes, wherein the circular DNA does not have an origin of replication. Preferably, the circular DNA does not have an origin of replication of either bacterial or eukaryotic origin.
Within yet other aspects of the present invention, recombinant adeno-associated cap (xe2x88x92) viruses are provided, comprising, 5xe2x80x2 AAV ITR, a first site specific recombination locus, the rep genes, a second site specific recombination locus which is capable of recombining with said first site specific recombination locus, and a 3xe2x80x2 AAV ITR, with the proviso that the recombinant adeno-associated cap (xe2x88x92) virus does not contain any functional cap genes. Within further embodiments, the recombinant adeno-associated cap(xe2x88x92) virus further comprises poly(A) sequence. Within related aspects, plasmids are provided which comprise a DNA sequence of such recombinant adeno-associated viruses.